Phenylethylamine derivatives and their use in the treatment of melanoma

ABSTRACT

Novel mono- and dihydroxy phenylethylamine derivatives useful in treating melanoma are provided having the formulae (Ia, Ib or Ic). In the above formulae, R a  is hydrogen or —COOR b , R b  is hydrogen or C 1-6  alkyl; R e  and R e  independently represent hydrogen and hydroxy, R f  is hydrogen, C 1–4? alkyl or halogen, X is —CHOH—, —CH 2 -oxygen or sulphur, m is zero or 1, W is oxygen or sulphur, and —ODrug, —NHDrug and —N(Drug) 2  each represent a residue of a therapeutically active agent. The above compounds are prodrugs which are inactive until metabolised by enzymes expressed by host melanoma cells. The invention allows a greater amount of active agent to be used while reducing systemic side effects

BACKGROUND TO THE INVENTION

It has recently been shown that tumours may be treated selectivelythrough the use of prodrugs, these prodrugs being inactive untilmetabolised by enzymes expressed by the host. This method of treatingcancers is promising, as it allows a greater amount of active agent tobe used while reducing systemic side effects.

This type of treatment has been proposed as having potential for thetreatment of malignant melanoma, in particular when utilising prodrugswhich are effectively recognised by the enzyme tyrosinase. Whentyrosinase acts upon the prodrug, an active antitumour agent isreleased.

In adults, tyrosinase is specifically produced in melanocytes, cellswhich are responsible for pigmentation. When a patient suffers frommelanoma the cancerous cells display a heightened level of tyrosinase.Thus, by employing a prodrug which is specifically recognised bytyrosinase, the prodrug can show greater selectivity for the cancerouscells.

Thus, our International Application No. PCT/GB97/03433 discloses novelprodrugs and assay reagents which are useful as therapeutic agents.These prodrugs and assay reagents were described as being substrates fortyrosinase and of being capable of releasing a therapeutically activeagent at a desired location. In particular, a compound called “ProdrugA” is disclosed. Prodrug A has certain drawbacks that make it less thanideal as viable drug for use in the treatment of melanoma. Thus, ProdrugA, although being a substrate for tyrosinase, is also a substrate forother enzymes present in the body. Furthermore, tests have shown ProdrugA to be relatively unstable in solution.

Hong et al. in Journal of Medicinal Chemistry, 1973, Vol. 16, No. 2,p139–147 disclose naturally-occurring 6-ureidopurines and thenucleosides. These compounds show cytokinin and growth inhibitoryactivity, but have not been shown to have activity on melanoma cells.

U.S. Pat. No. 4,115,539 relates to tyrosine derivatives which may becoupled with other compounds to form derivatives receptive to tagging,e.g. by radiolabeling. The derivatives are useful in detecting compoundsof interest in nano- or even pico-molar amounts. In one such compound,tyrosine is coupled to digoxin via a carbamate linker. The disclosedcompounds are stated to be useful in analysis techniques, but are notdisclosed to have any therapeutic applications, far less to be potentialcandidates for prodrug treatment of melanoma.

European Application No. 0 583 552 relates to conjugates of chlorophylland bacteriochlorophyll which are useful as photosensitizers in thediagnosis and therapy of tumours. The chlorophyll andbacteriochlorophyll moieties are conjugated with cell-specific ligandssuch as hormones, growth factors or tumour-specific antibodies tofacilitate targeting the tumour site.

It is an aim of the present invention to provide improved prodrugs foruse in the treatment of melanoma. It is a further object of theinvention to provide prodrugs which avoid problems associated with priorart prodrugs, specifically which show improved stability and increasedselectivity. Thus, it has been found that compared to Prodrug A,compounds which incorporate a urea linkage are advantageous. Prodrug Ahas a carbamate linkage between the therapeutically active entity andthe entity that is recognised by the tyrosinase enzyme. Further, a classof compounds which have only a single hydroxy substituent in the paraposition have been found to be excellent substrates for tyrosinaseenzymes, and to have high specificity.

SUMMARY OF THE INVENTION

According to one aspect of the invention, there are provided compoundshaving the formula (Ia), (Ib) or (Ic), or a pharmaceutically acceptablesalt thereof:

wherein R^(a) is hydrogen or —COOR^(b), R^(b) is hydrogen or C₁₋₆ alkyl;R^(e) and R^(e) independently represent hydrogen and hydroxy, R^(f)ishydrogen, C₁₋₄ alkyl or halogen, X is —CHOH—, —CH₂— oxygen or sulphur, mis zero or 1, W is oxygen or sulphur, and —ODrug, —NHDrug and —N(Drug)₂each represent a residue of a therapeutically active agent with theprovisos (1) that where said compound has general formula (Ia) wherein mis 0, W represents oxygen R^(d) is hydrogen and R^(e) is hydroxyl in thepara position, and R^(a) represents —COOMe, —ODrug does not represent aresidue of digoxin, and (2) that where said compound has general formula(Ib) wherein m is zero, W represents oxygen and R^(d) is hydrogen, R^(e)is hydroxyl in the para position.

According to a preferred aspect one of the invention, there are providedcompounds having the formulas (IIa), (IIb) or (IIc) or pharmaceuticallyacceptable salts thereof:

wherein R^(a) is hydrogen or —COOR^(b), and R^(b) is hydrogen or C₁₋₆alkyl; W is oxygen or sulphur, and —ODrug, —NHDrug and —N(Drug)₂represent a residue of a therapeutically active agent, with the provisothat where said compound has general formula (Ia) and R^(b) representsmethyl, —ODrug does not represent a residue of digoxin.

The invention also compounds having the general formulae (IIIa) and(IIIb) or pharmaceutically acceptable salts thereof.

wherein R^(a) is hydrogen or —COOR^(b), and R^(b) is hydrogen or C₁₋₆alkyl; —NHDrug and —N(Drug)₂ represent a residue of a therapeuticallyactive agent; and R^(d) and R^(e) independently represent hydrogen orhydroxy.

In a preferred embodiments of compounds of Formulae Ia, Ib, Ic, IIIa andIIIb, only one of R^(d) and R^(e) is hydrogen, with the remainingpositions on the phenyl ring remaining unsubstituted. It is particularlypreferable for the single hydroxy group to be present in the paraposition.

Further preferred classes of compounds of the invention are wherein:

-   -   R^(b) is hydrogen;    -   R^(b) is methyl;    -   R^(a) is hydrogen;    -   W represents oxygen.

It will be appreciated that compounds of the invention can formpharmaceutically acceptable salts, such as acid addition salts. Examplesof suitable acids for salt formation include hydrochloric, sulfuric,phosphoric, acetic, citric, oxalic, malonic, salicylic, malic, fumaric,succinic, ascorbic, maleic, methanesulfonic and other mineral andcarboxylic acids well known in the art.

Methods of preparation of acid addition salts will be apparent to thoseskilled in the art. For example, the salts may be prepared by contactingthe free base form with a sufficient amount of the desired acid toproduce a salt in the conventional manner. The free base forms may beregenerated by treating the salt with a suitabl dilute aqueous basesolution, such as dilute aqueous sodium hydroxide, potassium carbonate,ammonia or sodium bicarbonate. The free base forms differ from theirrespective salt forms somewhat in certain physical properties, such assolubility in polar solvents, but the acid and base forms are otherwiseequivalent to their respective free base forms for the purposes of theinvention.

All such acid and base salts are intended to be pharmaceuticallyacceptable salts within the scope of the invention and all acid and basesalts are considered equivalent to the free forms of the correspondingcompounds for the purposes of the invention.

The compounds of the invention may preferably be derived from the classof therapeutically active agents termed “mustards”. Such compoundstypically contain haloalkylamine e.g. bis(chloroethyl)amine groups.Mustards are able to act as intercalating agents, and cross-link strandsof DNA. In order to do so, the nitrogen lone pair first causes achloride ion to leave, forming a three-membered ring containing atertiary nitrogen atom. DNA then acts as a nucleophile, opening the ringand forming an entity believed to incorporate the structure:

This process is repeated, with the result being that the mustard acts asa “claw”, reacting with the DNA at two points (one on each strand), andpreventing the division of the DNA.

The compounds of the invention in this category (mustards) may contain—NHDrug, —ODrug and —N(Drug)₂ groups represented by the formulae:

Particularly preferred compounds of the invention have the followingformulae, known as “UPD”, “2.1.1” and “1.1.2” respectively:

Other examples of compounds according to the invention have residues oftaxol, gemcitabine or daunomycin in place of mustard groups discussedabove. Thus the residue of the therapeutically active agent maypreferably be selected from —O(Taxol), —O(Gemcitabine) and—NH(Daunomycin).

It is further within the scope of the invention to provide the use ofcompounds having the general formula Ia, Ib, Ic, IIa and IIb in themanufacture of pharmaceutical compositions for the treatment ofmelanoma.

Such pharmaceutical compositions may include any of the customaryexcipients used in formulating dosage forms. Suitable dosage formsinclude tablets, capsules, coated pills, injectable solutions andsuspensions.

GENERAL SYNTHETIC METHODS

The following section described general synthetic procedures forproducing compounds according to the invention.

Synthesis of Phenyl Mustard Prodrugs

The general protocol for the synthesis of the various prodrugs was basedon the premise of forming a reactive carbonate that was prone tonucleophilic attack by a primary or secondary amine. Prodrugs with acarbamate linkage were synthesised as previously reported¹, and requiredaccess to the reactive carbonate intermediate 6. Facile preparation ofp-hydroxyphenyl mustard hydrochloride 5 was achieved by treatingbenzyloxyaniline 2 with ethylene oxide to give diol 3.² Conversion tothe bis-chloroethyl amino compound 4 was facilitated using methanesulfonyl in anhydrous pyridine.³ Subsequent formation of thehydrochloride salt and benzyl cleavage by hydrogenation gave the desiredp-hydroxyphenyl mustard hydrochloride 5. Preparation of the carbonate 6,ready for coupling with primary and secondary amines, was achieved byheating 5 at reflux in toluene in the presence of p-nitrophenylchloroformate

Scheme 2: synthesis of the reactive carbonate 6. Reagents: (a)triethylamine, ethylene oxide, 88%, (b) mesyl chloride, pyridine, 59%,(c) HCl_((g)) then H₂ Pd/C, 51%, (d) p-nitrophenylchloroformat,triethylamine, 64%.

Coupling of carbonate 6 with various primary amines proceeded smoothlyin anhydrous dimethylformamide to give the corresponding prodrugs ingood to excellent yield (Table 1).

TABLE 1 phenyl mustard prodrugs aff rded from the reaction betweenvarious amines and carbonat 6. Entry Amine Carbonate Prodrug Yield 1

(6)

 (9)47%(10)73% (7) R = H (9) R = H (8) R = OH (10) R = OH 2

(6)

62% (11) (12) 3

(6)

(15)52%(16)54% (13) R = H (15) R = H (14) R = CO₂Me (16) R = CO₂Me 4

(6)

44% (17) (18) 5

(6)

(21)27%(22)39% (19) X = O (21) X = O (20) X = S (22) X = S

Since prodrug 12 was derived from an excellent tyrosinase substrate,tyrosine methyl ester, a thiocarbonate derivative was also prepared.Initial attempts at functional group interconversion, using Lawesson'sreagent⁴ were disappointing, and consequently an alternative synthesiswas adopted.

p-Hydroxyphenyl hydrochloride mustard 5 was treated withpentafluorophenylchlorothionoformate 23 to give thiocarbonate 24, whichwas converted to prodrug 25 by treatment with tyrosine methyl ester(Scheme 3).

Synthesis of bis-(2-chloroethyl)amine Urea Mustards

For this series of prodrugs two synthetic approaches were examined. Theinitial protocol mirrored that used for the formation of the phenolmustard prodrugs and involved the synthesis of the p-nitrophenolcarbamate 27 (Scheme 4).

Synthesis of the p-nitrophenyl carbamate 27 was easily achieved byreacting p-nitrophenyl chloroformate with bis-(2-chloroethyl)aminehydrochloride 26. Mustard 27 was then coupled to primary and secondaryamines as before (Scheme 5). In thes cases, the p-nitrophenol by-producthad to be removed from the prodrugs using silica gel columnchromatography.

In addition, with the aim of minimising purification procedures, twoone-pot methodologies were developed. The one-pot strategies relied uponin situ formation of a reactive intermediate which, upon reaction with aprimary or secondary amin, would afford the prodrug together with avolatile by-product. The one-pot rationale and formation of prodrugs28–30 can be seen in scheme 5.

In all cases, the one-pot approach afforded higher yields than employingthe p-nitrophenylchloroformate method. Therefore, when combined witheasier purification, the one pot approach is, without doubt, a superiormethod for prodrug synthesis.

The final class of compounds synthesised and tested by oximetry were thdaunomycin-based prodrugs. These were easily obtained using a similartwo pot reaction scheme, via the reactive carbamate 31. Addition ofdaunomycin then afforded the urea linked prodrug 32 (Scheme 6).

EXAMPLES

The synthesis of preferred compounds of the invention is illustrated inthe following examples.

General Experimental

All NMR spectra were recorded on a Bruker WM250, Bruker AC250, BrukerAvance DPX 250, Bruker AMX400 or Jeol AX400 spectrometer, using CHCl₃asan internal standard unless stated otherwise (7.26 ppm for ¹H NMR, 77.0ppm for ¹³C NMR). ¹³C spectra were recorded using DistortionlessEnhancement by Polarisation Transfer. Mass spectra were recorded on aFisons VG Autospec. Infra red spectra were recorded on a Perkin-ElmerParagon 1000 F.T.-I.R. spectrometer. Optical activities were determinedusing a Perkin-Elmer 341 polarimeter. Melting points were determinedusing an Electrothemal digital melting point apparatus, and areuncorrected. Scanning oximetry was performed using a YSI model 5300biological oxygen monitor.

Unless stated otherwise, all chemicals and materials were obtained fromthe Sigma-Aldrich Chemical Company, the B.D.H. Merk Chemical Company orLancaster Chemicals and were, used as received. Silica gel for columnchromatography was obtained from Merck, with a pore diameter of 6 nm.Alumina column chromatography was performed using 150 mesh neutralaluminium oxide, obtained from the Aldrich Chemical Company. Silica andalumina thin layer chromatography was performed on pre-coated aluminiumsheets, with a 0.2 mm thickness. Anhydrous solvents were purchased andused as received. Mushroom tyrosinase (3,520 units/mg) was used at aconcentration of 300 units/mL in 0.1 M phosphate buffered saline (pH7.4).

Example 1

Synthesis of UPD(4-di(2-chloroethyl)aminoanilino-4-hydroxyphenethylamino-methanone)

UPD was synthesised by the procedure shown in Scheme 1. Tyramine (101)(10 g, 73 mmol), and nitrophenyl isocyanate (102) (11.98 g, 73 mmol)were dissolved in anhydrous pyridine (5 mL) and stirred at roomtemperature for 6 hours. The mixture was concentrated under vacuum(residual Py azetroped using toluene) to give4-hydroxyphenethylamino4-nitroanilinmethanone (103) in quantitativeyield by TLC (CH₂Cl₂) and ¹H NMR.

(103) (10 g, 36.9 mmol), was dissolved in hot abs EtOH (250 mL) andallowed to cool before 5 mol % of 10% Pd on carbon was added. The flaskwas evacuated and H₂ was introduced under atmospheric pressure. Themixture was stirred for 12 hours, filtered through celite andconcentrated under vacuum to give4-aminoanilino-4-hydroxyphenethyl-aminomethanone (104) as an off-whitesolid in 100% yield.

(104) (5 g, 18,42 mmol) was dissolved in a solution of H₂O/MeOH/conc.AcOH (100 mL/10 mL/10 mL) and stirred at room temperature. To thissolution cold ethylene oxide (10 mol/eq) was added slowly. The mixturewas stirred at room temperature for 6 hours, and a further 10 mol/eq ofethylene oxide was added. This was repeated until an off-whiteprecipitate formed. The precipitate was filtered off and recrystallised(hot EtOAc) to give4-di(hydroxyethyl)aminoalinino-4-hydroxyphenethyl-aminomethanone (105)as an off-white solid (70% yield).

(105) (2.8 g, 7.8 mmol), CH₂Cl₂ (4 mL), pyridine (6 mL) were cooled andthionyl chloride (2.64 mL) was added. The mixture was heated at refluxfor 1 hour, allowed to cool and diluted with CH₂Cl₂ (200 mL). Themixture was washed with water (2×200 mL), th organics were dried overMgSO₄ and reduced under vacuum to give a brown oily solid (see compound106 (as identified by ¹H NMR and MS)).

The oily solid was then taken up in hot EtOAc/Ether (100 mL/100 mL) andallowed to cool. Once cool, HCl gas was passed through the solution togive an off-white precipitate. The precipitate was filtered off andconcentrated under vacuum to give (UPD) as a light brown honeycombsolid, which was recrystallised (hot EtOAc) to afford >99.5 purity byHPLC.

Example 2

Synthesis of 1.1.2(bis-(2-chloroethyl)amino-4-hydroxyphenylamino-methanone): One PotMethod

The synthesis of 1.1.2 is shown in Scheme 2. Bis-(2-Chloroethyl)aminehydrochlorid (107) (200 mg, 1.1 mmol) was dissolved in dichloromethane(15 mL), and triethylamine (0.45 mL, 3,3 mmol) was added. The mixturewas stirred for 5 minutes at ambient temperature and ethyl chloroformate(108) or methyl chlorothioformate (1.32 mmol) was added. The mixture wasstirred until no bis-(2-chloroethyl)amine remained by TLC (ethylacetate) (109).

Tyramine (110) (300 mg, 2,2 mmol) was added and the mixture was heatedund r reflux for 4 hours. The mixture was allowed to cool, purified bydry flash column chromatography (silica gel, dichloromethane 200 cm³)and concentrated in vacuo to yield prodrug 1.1.2 as an orange/brown oil(234 mg, 67% for methyl chlorothioformate and 229 mg, 64% for ethylchloroformate).

Example 3

Synthesis of 2.1.1 ((R)-[2′-amin-3-(4″-hydroxyphenyl)propionic acidmethyl ester]-carbamic acid p-(bis-2-chloroethylamino) ph nyl est r)

2.1.1 was synthesised by the following procedure, as shown in Scheme 3.4-benzyloxyaniline hydrobromide (111) (11.8 g, 0.05 mol) was suspendedin glacial acetic acid (70 cm³) and water (70 cm³) and cooled to 0° C.Ethylene oxide (17.64 g, 20 cm³, 0.4 mol) was then added, in 1 cm³portions, the solution allowed to warm to room temperature and stirreduntil no starting material remained by TLC. Additional ethylene oxidewas added as required in order to drive the reaction to completion.After this time, the solution was concentrated in vacuo (T<60° C.) togive a red/brown syrup, which was re-dissolved in chloroform (100 cm³).This solution was washed with water (50 cm³) and saturated sodiumbicarbonate solution (50 cm³), dried (magnesium sulfate), filtered andconcentrated in vacuo. Recrystallisation (toluene/hexane) gave the diolBenzyl-p-(bis-2-hydroxyethylamino) phenyl ether (112) as a pale creampowder (12.55 g, 88%).

Phosphorous Pentachloride Method:

(112) (5.8 g, 0.02 mol) was dissolved in chloroform (30 cm³) and cooledto 0° C. Phosphorous pentachloride (12.5 g, 0.06 mol) was slowly added,the mixture warmed to room temperature and left to stand overnight. Theresultant black mixture was then refluxed for one hour, after which timeit was cooled to room temperature, poured onto crushed ice (20 cm³),stirred vigorously and poured onto an additional portion of crushed ice(20 cm³). The organic layers were separated, washed with saturatedbicarbonate solution (2×50 cm³) and dried (magnesium sulfate). Afterfiltration and concentration in vacuo, the resultant black oil waspurified by eluting through a short pad of silica with dichloromethane.Recrystallisation (dichloromethane:hexane) gave the dichloride,benzyl-p-(bis-2-chloroethylamino)phenyl ether (113), as white plates(3.33 g, 52%).

Mesyl Chloride Method:

(112) (2.0 g, 7.2 mmol) was dissolved in anhydrous pyridine (11 cm³) andcooled to 0° C. Mesyl chloride (28.8 mmol, 2.23 cm³) was added and thesolution stirred at 2–4° C. for 20 minutes, followed by heating at 80°C. for 30 minutes. Ethyl acetate (30 cm³) and wat r (30 cm³) were thenadded, the organic fraction collected, dried (magnesium sulfate),filtered and concentrated in vacuo. Column chromatography (silica,dichloromethane) gave the dichloride (113) (1.38 g, 59%) as a whitepowder.

Hydrogen chloride gas was bubbled through a solution of (113) (3.3 g,0.01 mol) in methanol (35 cm³), until complete dissolution occurred.

Filtration and concentration in vacuo gave the hydrochloride salt as awhite powder, which was immediately re-suspended in ethanol (40 cm³)containing 10% palladium on carbon (0.17 g). The suspension was stirredunder an atmosphere of hydrogen until no starting material remained(TLC). The suspension was then filtered over celite and concentrated invacuo to give p-(bis-2-chloroethylamino)phenol hydrochloride (114) as awhite solid (1.37 g, 51%).

(114) (1.35 g, 0.057 mmol) and triethylamine (1.17 g, 1.61 cm³, 0.114mol) in toluene (15 cm³) was slowly added, over 15 minutes, to arefluxing solution of p-nitrophenyl chloroformate (115) (1 g, 0.005 mol)in toluene (15 cm³) and the mixture refluxed for one hour. After thistime, the reaction was cooled, concentrated in vacuo and purified bycolumn chromatography (silica, dichloromethane) to give the diester,carbonic acid-p-(bis-2-chloroethylamino)phenyl ester-p-nitrophenyl ester(PNMC) (116) as a yellow oil (1.27 g, 64%) which solidified uponstanding.

To a solution of nitrophenyl carbonate (116) (1.35 g, 0.057 mmol) inchloroform (1 cm³) was added L-tyrosine methyl ester (117) (0.127 g,0.65 mmol) and the mixture refluxed for four hours. After this time, thereaction was cooled and concentrated in vacuo. Column chromatographygave prodrug 2.1.1 as a colourless oil (0.085 g, 62%).

Example 4

Synthesis of 1.1.5(3-Acetyl-3,5,12-trihydroxy-1-[5-hydroxy-4-(4-hydroxyphenylaminocarbonylamino)-6-methylperhydro-2-pyranoloxy]10-methoxy-(1S,3S)-1,2,3,4,6,11-hexaydro-6,11-napthacnedione)

The synthesis of 1.1.5 is shown in Scheme 4. Tyramine (119) (1 g, 7.3mmol) and p-nitrophenylchloroformate (120) (1.4 g, 7.3 mmol) weredissolved in anhydrous dichloromethane and heated under reflux for 2hours. The reaction mixture was allowed to cool, concentrated in vacuoand purified by dry flash column chromatography (silica, dichloromethaneand then ethylacetate) to afford carbamate (121) as a pale yellow solid(2.2 g, 97%); m.p. 157–159° C.; ν_(max) (KBr disc) 400, 1658, 1440,1380/cm⁻¹; δ ¹H (400 MHz, CDCl3) 2.74 (2H, t, J 7.0 Hz, CH2Ar), 3.4 (2H,t, J7.0 Hz, CH2) 6.76 (2H, d, J8 Hz, 2×ArH), 8.24 (2H, d, J9.2 Hz, ArH);δ ¹³C(100 MHz, CDCl₃); 36.4 (CH₂), 44.2 (CH₂), 116.6 (2×CH), 123.7(2×CH), 26.4(2×CH), 131.2 (2×CH), 131.3 (C) 146.9 (C), 156.0 (C), 157.4(C), 158.1 (C). m/z (Cl) 162 (25%), 139 (10), 107 (100) 65 (15).

Daunomycin (122) (20 mg, 0.038 mmol) and carbamate (121) (15 mg, 0.49mmol) were dissolved in dimethylformamide (1 mL) anddiisopropylethylamine (7.5 μL, 0.042 mmol) was added. The flask waswrapped in tinfoil to exclude light and the mixture was stirred for 3hours. Diethyl ether (5 mL) was added to give a red precipitate. Theprecipitate was collected by filtering across a cotton wool plug. Thesolid was then washed off the cotton wool using methanol (5 mL) andconcentrated in vacuo to yield prodrug 1.1.5 as a red oily solid (12 mg,46%). ν_(max)3400, 2720, 1740, 1750, 1690, 1520, 1435, 1147/cm⁻¹; δ¹H(400) MHz, CD₃OD) 1.25 (2H, d, J 4.5 Hz, CH₂), 1.32–1.4 (5H, m, 5′CH₃and CH ₂CCOH₃), 1.72–1.89 (2H, m 2′CH₂), 2.35 (3H, s, COCH₃), 2.55(2H, t, J 3.72 Hz, ArCH₂ CH ₂N), 3.54–3.55 (1H, m, 4′CHOH), 3.71 (1H, m,C(CHO)CH₂), 3.83 (3H, s, ArOCH₃), 3.92 (1H, brd, 1′CH), 4.25 (1H, q, J3.8 Hz, 5′CH), 5.31–5.33 (1H, m, 3′CH), 6.58 (2H, d, J 5.6 Hz, 2×ArH),6.91 (2H, d, J 5.6 Hz, 2×ArH), 7,27 (1H, t, J 3.3 Hz, ArH), 7.55–7.57(2H, m, 2×ArH; δ¹³C (100 MHz, CD₃OD) 25.1 (CH₃), 31.2 (CH), 32.0 (CH₂),33.9 (CH₂), 37.0 (CH₂), 43.2 (CH₂, 44.2 (CH₂), 47.6 (CH) 56.2 (CH), 57.4(CH), 69.1 (CH), 74.3 (CH₃) 77.9 (C), 80.0 (CH3) 102.7 (CH), 131.1(3×CH), 131.8 (CH), 135.9 (2×C), 136.2 (2×C), 136.3 (2×C), 137.4 (2×CH),156.4 (C), 157.2 (C), 157.7 (2×C), 160.6 (2×C), 187.6 (C), 187.9 (C),214.1 (C); m/z (Cl) 383 (20%), 363 (100), 347 (15), 293 [urea linkedtyramine to 4-OH-5-Me-hexose(15)], 174 (10), 138 (10), 107 (10), 74(10).

Examples 5

Carbonic acid-p-(bis-2-chloroethylamino)phenyl ester-p-nitrophenyl ester(PNMC) (6).

Benzyl-p-(bis-2-hydroxyethylamino)phenyl ether (3).

4-Benzyloxyaniline hydrobromide (11.8 g, 0.05 mol) was suspended inglacial acetic acid (70 mL) and water (70 mL) and cooled to 0° C.Ethylene oxide (17.64 g, 20 cm³, 0.4 mol) was then added, in 1 mLportions, the solution allowed to warm to room temperature and stirreduntil no starting material remained by TLC. Additional ethylene oxidewas added as required in order to drive the reaction to completion.After this time, the solution was concentrated in vacuo (T<60° C.) togive a red/brown syrup, which was re-dissolved in chloroform (100 mL).This solution was washed with water (50 mL) and saturated sodiumbicarbonate solution (50 mL), dried (magnesium sulfate), filtered andconcentrated in vacuo. Re-crystallisation (toluene/hexane) gave the diolas a pale cream powder (12.55 g, 88%); m.p. 96–7° C. (lit.⁵ 93–4° C.);R_(f) (silica, ethyl acetate) 0.3; δ ¹H (400 MHz, CDCl₃) 3.71–3.82 (8H,br m. 4×CH₂), 6.79 (2H, br d., J 8.6 Hz, PhCH₂), 7.39 (2H, br d., J 8.6Hz, Ph), 7.41–7.55 (7H, m, Ar).

Benzyl-p-(bis-2-chloroethylamino)phenyl ether (4).

The bis-(hydroxyethylamino)phenyl ether 3 (2.0 g, 7.2 mmol) wasdissolved in anhydrous pyridine (11 mL) and cooled to 0° C. Mesylchloride (28.8 mmol, 2.23 mL) was added and the solution stirred at 2–4°C. for 20 minutes, followed by heating at 80° C. for 30 minutes. Ethylacetate (30 mL) and water (30 mL) were then added, the organic fractioncollected, dried (magnesium sulfate), filtered and concentrated invacuo. Column chromatography (silica gel, dichloromethane) gave thedichloride (1.38 g, 59%) as a white powder, m.p. 108–09° C. (lit.⁶105–06° C.); R_(f) 0.7 (silica, dichloromethane); δ ¹H (400 MHz, CDCl₃)3.16–3.27 (8H, m, 4×CH₂), 5.00 (2H, s, PhCH₂), 6.73 (2H, d, J 8.6 Hz,Ph), 6.92 (2H, d, J 8.6 Hz, Ar), 7.29–7.44 (5H, m, Ar).

p-(Bis-2-chloroethylamino)phenol hydrochloride (5).

Hydrogen chloride gas was bubbled through a solution of thebis-(chloroethylamino)phenyl ether 4 (3.3 g, 0.01 mol) in methanol (35mL), until complete dissolution occurred. Filtration and concentrationin vacuo gave the hydrochloride salt as a white powder {m.p. 140–41° C.(lit⁷ 135–36° C.)}, which was immediately re-suspended in ethanol (40mL) containing 10% palladium on carbon (0.17 g). The suspension wasstirred under an atmosphere of hydrogen until no starting materialremained (TLC). The suspension was then filtered over celite andconcentrated in vacuo to give the phenol hydrochloride as a white solid(1.37 g, 51%); m.p. 176–78° C. (170–73° C.); δ ¹H (400 MHz, CDCl₃, freeamine) 3.52–3.63 (8H, m, 4×CH₂), 6.57 (2H, d, J 9.0 Hz, Ar), 6.67 (2H,d, J 9.0 Hz, Ar).

Carbonic acid-p-(bis-2-chloroethylamino)phenyl ester-p-nitrophenyl ester(PNMC) (6).

The bis-chloroethylamino hydrochloride salt 5 (1.35 g, 5.79 mmol) andtriethylamine (1.17 g, 1.61 mL, 11.4 mmol) in toluene (15 mL) was slowlyadded, over 15 minutes, to a refluxing solution ofp-nitrophenylchloroformate (1 g, 4.96 mmol) in toluene (15 mL) and themixture was heated under reflux for one hour. After this time, thereaction was cooled, concentrated in vacuo and purified by columnchromatography (silica gel, dichloromethane) to give the diester as ayellow oil (1.27 g, 64%) which solidified upon standing; m.p. 97–99° C.;R_(f) 0.77 (silica, dichloromethane); ν_(max) (KBr disc) 1767,1615,1594, 1512, 1347, 1180, 814/cm⁻¹; δ ¹H (400 MHz, CDCl₃) 3.61–3.68 (4H,m, 2×CH₂), 3.71–3.88 (4H, m, 2×CH₂), 6.70 (2H, d, J 9.2 Hz, Ar), 7.16(2H, d, J 9.2 Hz, Ar), 7.47 (2H, d, J 9.1 Hz, Ar), 8.30 (2H, d, J 9.1Hz, Ar);

^(□)C (100 MHz, CDCl₃) 40.2 (CH₂), 53.6 (CH₂), 112.4 (CH), 121.7 (CH),121.8 (CH), 125.3 (CH), 140.5 (C), 141.3 (C), 142.7 (C), 143.1 (C),153.6 (C); (Cl: found: 399.0525 [M+H]⁺. C₁₇H₁₆Cl₂N₂O₅ [M+H]⁺, requires399.0514); m/z (Cl) (399 [M+H]⁺, 75%), 348 (100), 120 (20), 63 (15).

Example 6

{2′-(4″-Hydroxyphenyl)ethyl} carbamic acidp-(bis-2-chloroethylamino)phenyl ster (9).

To a solution of the nitrophenyl carbonate 6 (0.20 g, 0.57 mmol) inchloroform (1 mL) was added tyramine hydrochloride (0.096 g, 0.50 mmol)and triethylamine (0.50 g, 0.08 mL, 0.50 mmol) and the mixture washeated under reflux for four hours. After this time, the reaction wascooled, concentrated in vacuo and purified by column chromatography(silica gel, dichloromethane:ethyl acetate 95:5) to give the carbamate 9as a colourless oil (0.046 mg, 47%); R_(f) 0.29 (silica,dichloromethane:ethyl acetate 95:5); □_(max) (KBr disc) 3336, 1718,1612, 1507, 1336, 1219/cm⁻¹; δ ¹H (400 MHz, CDCl₃) 2.80 (2H, t, J 6.9Hz, PhCH ₂), 3.47 (2H, apparent dd, J 6.9 Hz, 6.0 Hz, CH ₂NH), 3.61 (2H,d, J 6.0 Hz, 2×NCH), 3.68 (4H, d, J 6.0 Hz, 2×CH₂Cl), 6.67 (1H, br t, J6.0 Hz, NH), 6.67 (2H, d, J 9.0 Hz, Ar), 6.77 (2H, d, J 8.4 Hz, Ar),6.99 (2H, d, J 9.0 Hz, Ar), 7.03 (2H, d, J 8.4 Hz, Ar);

¹³C (100 MHz, CDCl₃); 35.3 (CH₂), 40.5 (CH₂), 42.8 (CH₂), 54.3 (CH₂),113.4 (CH), 115.8 (CH), 123.1 (CH), 130.2 (CH), 130.7 (C), 143.6 (C),154.7(C), 155.8 (C and CO); (Cl: found: 397.1085 [M+H]⁺. C₁₉H₂₂Cl₂N₂O₃requires 397.1085); m/z (Cl) (397 [M+H]⁺, 45%), 234 (100), 184 (95), 107(100).

Example 7

{2′-(3″,4″-Dihydroxyphenyl)-ethyl} carbamic acidp-(bis-2-chloroethylamino) phenyl ester (10).

A solution of the carbonate 6 (0.1 g 0.26 mmol), 3-hydroxytyraminehydrochloride (0.1 g, 0.53 mmol) and triethylamine (0.05 g, 0.07 mL, 0.5mmol) in anhydrous dimethylformamide (1.5 mL) was stirred for three daysat ambient temperature. After this time, the mixture was concentrated todryness in vacuo. Column chromatography (silica gel,dichloromethane:methanol 100:1→9:1 v/v) gave the carbamate 10 as acolourless viscous oil (0.08 g, 73%); R_(f) 0.45 (silica,dichloromethane:methanol, 9:1, v/v); ν_(max) (KBr disc) 3421, 1718,1653, 1507, 1218 cm⁻¹; δ ¹H(400 MHz, CDCl₃) 2.60 (2H, br t, J 6.3 Hz,PhCH ₂), 3.32 (2H, br q, J 6.3 Hz, CH ₂NH), 3.48 (4H, d, J 6.2 Hz,2×NCH₂), 3.55 (4H, d, J 6.2 Hz, 2×CH₂Cl), 5.17 (1H, br t, J 6.3 Hz, NH),6.50 (2H, d, J 7.9 Hz, Ar), 6.58 (2H, s, Ar), 6.64 (1H, d, J 7.7 Hz,Ar), 6.88 (2H, d, J 7.9 Hz, Ar);

^(□)C (100 MHz, CDCl₃) 35.3 (CH₂), 40.7 (CH₂), 42.7 (CH₂), 53.9 (CH₂),112.8 (CH), 115.6 (CH), 116.8 (CH), 120.9 (CH), 123.0 (CH), 130.9 (C),142.5 (C), 143.0 (C), 144.1 (C), 144.4 (C), 156.1 (C and CO); (Cl:found: [M+H]⁺, 413.1045. C₁₉H₂₂Cl₂N₂O₄ requires 413.1034); m/z (Cl) 413([M+H]⁺, 10%), 233 (50), 184 (100), 123 (35).

Example 8

(R)-[2′-Amino-3′-(4″-hydroxyphenyl)propionic acid methyl ester]-carbamicacid p-(bis-2-chloroethylamino) phenyl ester (12).

To a solution of the nitrophenyl carbonate 6 (0.20 g, 0.57 mmol) inchloroform (1 mL) was added L-tyrosine methyl ester (0.127 g, 0.65 mmol)and the mixture was heated under reflux for four hours. After this time,the reaction was cooled and concentrated in vacuo. Column chromatography(silica gel, dichloromethane:methanol 100:1) afforded carbamate 12 as acolourless oil (0.085 g, 62%); R_(f) 0.16 (silica, dichloromehane:ethylacetate 95:5); [α]_(D) ²⁰ +29.0° (c 0.9, chloroform); ν_(max) (KBr disc)3446, 1718, 1700, 1559, 1496, 1218/cm⁻¹; δ ¹H (400 MHz, CDCl₃) 3.03 (2H,dq, J 5.9 Hz, 14.2 Hz PhCH ₂), 3.54 (2H, d, J 6.2 Hz, 2×NCH₂), 3.68 (2H,d, J 6.0 Hz, 2×CH₂Cl), 3.69 (3H, s, Me), 4.58 (2H, br d, J 6.6 Hz, CH),5.41 (1H, d, J 8.0 Hz, NH), 6.61 (2H, d, J 9.0 Hz, Ar), 6.68 (2H, d, J8.4 Hz, Ar), 6.92 (2H, d, J 9.1 Hz, Ar), 6.94 (2H, d, J 9.1 Hz, Ar);

¹³C (100 MHz, CDCl₃); 37.3 (CH₂), 40.3 (CH₂), 52.4 (CH₃), 53.7 (CH₂),55.0 (CH), 112.6 (CH), 115.5 (CH), 122.7 (CH) 127.2 (C), 130.3 (CH),142.3 (C), 143.8 (C), 154.7 (C), 155.0 (C), 172.0 (C); (Cl: found:455.1148 [M+H]⁺. C₂₁H₂₄Cl₂N₂O₅ requires 455.1141); m/z (Cl) (455 [M+H]⁺,10%), 234 (15), 184 (35), 107 (100).

Example 9

3′,4′-Dihydroxybenzylamino-carbamic acid p-(bis-2-chloroethylamino)phenyl ester (15).

A solution of carbonate 6 (0.13 9, 0.32 mmol), 3,4-dihydroxybenzylaminehydrobromide (0.14 g, 0.65 mmol) and triethylamine (0.07 g, 0.09 mL,0.65 mmol) in anhydrous dimethylformamide (2 cm³) was stirred at roomtemperature for 72 hours. After this time, the mixture was concentratedto dryness in vacuo. Column chromatography (silica gel,dichloromethane:methanol 100:1→9:1 v/v) gave carbamate 15 as a whitepowder (0.07 g, 52%); R_(f) 0.29 (silica, dichloromethane: methanol,9:1, v/v); ν_(max) (KBr disc) 3322, 1689, 1616, 1507, 1427, 1281, 1215,1037/cm⁻¹; δ ¹H (400 MHz, CD₃OD) 3.65 (4H, d, J 6.2 Hz, 2×NCH₂), 3.71(4H, d, J 6.2 Hz, 2×CH₂Cl), 4.17 (2H, br d, J 7.3 Hz, CH₂), 6.64 (1H, brt, J 7.3 Hz, NH), 6.74–6.81 (5H, m, Ar), 6.96 (2H, d, J 7.4 Hz, Ar);

¹³C (100 MHz, CD₃OD) 41.6 (CH₂), 45.3 (CH₂), 54.6 (CH₂), 113.9 (CH),115.7 (CH), 116.2 (CH), 119.9 (CH), 123.7 (CH), 144.0 (C), 145.4 (C),145.6 (C), 146.3 (C), 158.0 (C and CO); (Cl: found: [M+H]⁺, 399.0870.C₁₈H₂₀Cl₂N₂O₄ requires 399.0878); m/z (Cl) ([M+H]⁺, 20%), 363 (10), 233(70), 184 (100).

Example 10

(R)-[1′-Amino-2′-(3,4′-hydroxyphenyl)ethanonic acid methylester]-carbamic acid p-(bis-2-chloroethylamino) phenyl ester (16).

(R)-3,4-Hydroxyphenylglycine methyl ester (14). To a stirred solution of3,4-hydroxyphenylglycine (0.5 g, 3.0 mmol) in 2,2-dimethoxypropane (30mL) was added concentrated hydrochloric acid (3 mL). After stirringovernight at 20° C., the mixture was concentrated to dryness in vacuo,and minimal methanol added to re-dissolve the residues. Diethyl ether(75 mL) was added and the resultant solid obtained by filtration.Re-dissolution in methanol (30 mL) and addition of triethylamine (0.3 g,0.4 mL, 3 mmol), followed by concentration in vacuo and columnchromatography (silica gel, dichloromethane:methanol 20:1 v/v) gave thefree amine 14 as a white powder in quantitative yield; m.p. 172–73° C.(lit.⁸ 178–80° C.); [α]_(D) ²⁰ −114.4° (c 0.25, 10% aq. hydrochloricacid) {lit.⁹ [α]_(D) ²⁰ −121.1° (c 1, aq. hydrochloric acid)}; R_(f)0.55 (silica, dichloromethane:methanol, 10:1, v/v); ν_(max) (KBr disc)3447, 1734, 1559, 1517, 1465, 1281, 1255, 1220, 1167/cm⁻¹; δ ¹H (400MHz, CDCl₃) 3.24 (2H, br s, NH₂), 3.60 (3H, s, Me), 4.46 (1H, br s.,CH), 6.70 (2H, d, J 8.6 Hz, Ar), 7.07 (2H, d, J 8.6 Hz, Ar);

¹³C (100 MHz, CDCl₃) 55.1 (CH), 61.3 (CH₃), 118.9 (CH), 131.7 (CH),134.0 (C), 161.0 (C), 178.2 (C); (Cl: found: [M+H]⁺, 182.0822. C₉H₁₁NO₃requires 182.0818); m/z (Cl) 182 ([M+H]⁺, 15%), 165 (50), 122 (100), 107(5).

(R)-[1′-Amino-2′-(3,4″-hydroxyphenyl)ethanonic acid methylester]-carbamic acid p-(bis-2-chloroethylamino) phenyl ester (16).

A solution of the carbonate 6 (0.13 g, 0.32 mmol) and the amino acidmethyl ester 14 (0.14 g, 0.65 mmol) in anhydrous dimethylformamide (2mL) was stirred at room temperature for 72 hours. After this time, themixture was concentrated to dryness in vacuo. Column chromatography(silica gel, dichloromethane:ethyl acetate, 95:5, v/v) gave carbamate 16as a colourless viscous oil (0.07 g, 54%); [α]_(D) ²⁰ −115.8° (c 0.85,chloroform); R_(f) 0.14 (silica, dichloromethane:ethyl acetate, 95:5,v/v); ν_(max) (KBr disc) 3384, 1718, 1612, 1506, 1437, 1350, 1218, 1174,1030/cm⁻¹; δ ¹H (400 MHz, CDCl₃) 3.17 (3H, s, OMe), 3.48 (4H, d, J 6.2Hz, 2×NCH₂), 3.57 (4H, d, J 6.2 Hz, 2×CH₂Cl), 5.23 (1H, d, J 7.0 Hz,NH), 6.08 (1H, d, J 7.0 Hz, CH), 6.52 (2H, d, J 8.0 Hz, Ar), 6.61 (2H,d, J 7.7 Hz, Ar), 6.90 (2H, d, J 7.7 Hz, Ar), 7.10 (2H, d, J 8.0 Hz,Ar);

¹³C (100 MHz, CDCl₃) 40.3 (CH₂), 52.9 (CH₃), 53.6 (CH₂), 57.4 (CH),112.5 (CH), 115.8 (CH), 122.6 (CH), 127.6 (C), 128.4 (CH), 142.1 (C),143.8 (C), 154.5 (C), 156.4 (C), 171.5 (C); (Cl: found: [M+H]⁺,441.0968. C₂₀H₂₂Cl₂N₂O₅ requires [M +H]⁺, 441.0984); m/z (Cl) 441([M+H]⁺, 10%), 277 (10), 233 (35), 184 (70), 147 (100).

Example 11

(R)-[1′-Amino-2′-hydroxy-2′-(4″-hydroxyphenyl)proplonic acid methylester]-carbamic acid p-(bis-2-chloroethylamino) phenyl ester (18).

A solution of the carbonate 6 (0.13 9, 0.32 mmol) and L-adrenaline (0.12g, 0.65 mmol) in anhydrous dimethylformamide (2 mL) was stirred at roomtemperature for 72 hours. After this time, the mixture was concentratedto dryness in vacuo. Column chromatography (silica gel,dichloromethane:methanol 100:1→9:1 v/v) gave carbamate 18 as a viscouscolourless oil (0.06 g, 44%); [α]_(D) ²⁰ −21.9° (c 0.95, chloroform);R_(f) 0.29 (silica, dichloromethane:methanol, 9:1, v/v); ν_(max) (KBrdisc) 3368, 1700, 1611, 1516, 1448, 1357, 121, 1219/cm⁻¹; δ ¹H (400 MHz,CDCl₃) 3.41 (2H, d, J 7.4 Hz, CH₂), 3.52 (3H, s, Me), 3.53 (4H, d, J 6.2Hz, 2×NCH₂), 3.64 (4H d, J 6.2 Hz, 2×CH₂Cl), 4.81 (1H, d, J 7.4 Hz, CH),6.57–6.92 (7H, m, Ar);

¹³C (100 MHz, CDCl₃) 34.7 (CH₃), 36.3 (CH₂), 40.4 (CH₂), 53.6 (CH₂),72.5 (CH), 112.5 (CH), 113.1 (CH), 115.2 (CH), 118.2 (CH), 122.7 (CH),133.5 (C), 142.5 (CH), 143.9 (C), 144.0 (C), 157.2 (C and CO); (Cl:found: [M+H]⁺, 443.1079. C₂₀H₂₄Cl₂N₂O₅ requires 443.1140); m/z (Cl) 443([M+H]₊, 20%), 425 (100), 234 (70),184 (100), 184 (85), 121 (20).

Example 12

{2′-(4″-Hydroxyphenyl)ethylamine) carbamic acidp-(bis-2-chloroethylamino) phenyl ester (21).

2-(4-Hydroxyphenoxy)-ethylamine hydrochloride (19).¹⁰

A solution of 4-hydroxyacetamide 34 (1.45 g, 8.8 mmol) in anhydroustetrahydrofuran (63 mL) was slowly added under an inert atmosphere to arefluxing suspension of lithium aluminium hydride (1M soln. in anhydroustetrahydrofuran, 32 mL) and the suspension refluxed for a further 12hours. After cooling, water was slowly added until hydrogen evolutionceased and the mixture concentrated in vacuo. After re-suspension inmethanol, the mixture was eluted through a short pad of silica withmethanol and the organic extracts concentrated in vacuo, re-dissolved inconcentrated hydrochloric acid and re-concentrated in vacuo to give theamine hydrochloride as a white powder (0.2 g, 12%); m.p. 170–71° C.(lit.¹⁰ 172–74° C. dec.); ¹H (250 MHz, D₂O) δ 3.25 (2H, t, J 5.2 Hz,CH₂), 4.08 (2H, t, J 5.2 Hz, CH₂), 6.73 (2H, d, J 9.1 Hz, Ar), 6.82 (2H,d, J 9.1 Hz, Ar).

2-[(4-hydroxyphenyl)thio]ethylamine hydrochloride (20).

A mixture of 4-hydroxythiophenol (2.48 g, 19.7 mmol) and2-methyl-2-oxazoline (1.67 g, 1.69 mL, 19.7 mmol) were refluxed (neat)under argon for two hours. Upon cooling, the crude sticky solid wasre-suspended in concentrated hydrochloric acid (aq) and refluxed for 12hours. The mixture was then poured into water (20 mL) and extracted withdiethyl ether (2×20 mL). The aqueous liquors were concentrated todryness and twice re-dissolved in water and re-concentrated.Re-crystallisation of the resultant solid (ethanolidiethyl ether) gavethe product as a cream solid (0.45 g, 11%); m.p. 130–31 ° C. (lit.¹¹128–29° C.); ¹H (400 MHz, D₂O) δ 3.07 (4H, br s, 2×CH₂), 6.87 (2H, d, J11.9 Hz, Ar), 7.41 (2H, d, J 11.9 Hz, Ar)

{2′-(4″-Hydroxyphenyl)ethylamine} carbamic acidp-(bis-2-chloroethylamino) phenyl ester (21).

A solution of 19 (0.2 g, 1 mmol), 6 (0.3 g, 0.75 mmol) and triethylamine(0.1 g, 0.14 mL, 1 mmol) in anhydrous chloroform (3 mL) was heated underreflux under an inert atmosphere for 4 hours and concentrated in vacuo.Column chromatography (silica gel, ethyl acetate:dichloromethane 1:19v/v) gave carbamate 21 as a colourless oil (0.11 g, 27%); R_(f) 0.26(silica, ethyl acetate:dichloromethane 1:19 v/v); ν_(max) (KBr disc)3358, 1713, 1612, 1506, 1452, 1337, 1217, 1109, 1068, 826/cm⁻¹; δ ¹H(250 MHz, CDCl₃) 3.47 (4H, d, J 7.8 Hz, 2×CH₂), 3.56 (2H, t, J 5.0 Hz,CH₂), 3.65 (4H, d, J 7.8 Hz, 2×CH₂), 3.91 (2H, t, J 5.0 Hz, CH₂). 5.55(1H, t, J 5.8 Hz, NH), 6.53 (2H, d, J 9.1 Hz, Ar), 6.67 (4H, d, J 1.4Hz, Ar), 6.90 (2H, d, J 9.1 Hz, Ar);

¹³C (62.8 MHz, CDCl₃) 40.7 (CH₂), 41.3 (CH₂), 54.1 (CH₂), 67.6 (CH₂),112.9 (CH), 115.9 (CH), 116.5 (CH), 123.2 (CH), 142.7 (C), 144.2 (C),150.7 (C), 152.6 (C), 156.2 (C and CO); (Cl: found: [M+H]⁺, 413.1049.C₁₉H₂₂N₂O₄ requires 413.1034); m/z (Cl) 413 ([M+H]⁺, 15%), 234 (70), 184(100), 135 (10), 110 (55), 65 (15).

Example 13

2′-[(4″-Hydroxyphenyl)thio]ethylamine} carbamic acidp-(bis-2-chloroethylamino) phenyl ester (22).

A solution of 20 (0.2 g, 1 mmol) and triethylamine (0.1 g, 0.14 mL, 1mmol) in anhydrous chloroform (3 mL) was brought to reflux and 6 (0.3 g,0.75 mmol) added. After refluxing under an inert atmosphere for 4 hoursand concentration in vacuo, column chromatography (silica gel, ethylacetate:dichloromethane 1:19 v/v) gave carbamate 22 as a colourless oil(0.16 g, 39%); R_(f) 0.34 (silica, ethyl acetate:dichloromethane 1:19v/v); ν_(max) (KBr disc) 3333, 1700, 1651, 1612, 1556, 1495, 1455, 1397,1335, 1266, 1218, 1182, 1110, 1048/cm⁻¹; δ ₁ ¹³H (250 MHz, CDCl₃) 2.88(2H, d, J 6.3 Hz CH₂), 3.34 (2H, q, J 6.3 Hz, CH₂), 3.51 (4H, d, J Hz,2×CH₂), 3.58 (4H, d, J Hz, 2×CH₂), 5.44 (1H, t, J 6.0 Hz, NH), 6.57 (2H,d, J 9.1 Hz, Ar), 6.64 (2H, d, J 11.7 Hz, Ar), 6.91 (2H, d, J 9.1 Hz,Ar), 7.20 (2H, d, J 11.7 Hz, Ar);

¹³C (62.8 MHz, CDCl₃) 36.1 (CH₂), 40.5 (CH₂), 40.7 (CH₂), 54.0 (CH₂),112.9 (CH), 116.7 (CH), 123.1 (CH), 126.4 (CH), 134.5 (CH), 142.6 (C),144.3 (C), 156.1 (C), 156.4 (C and CO).

Example 14

2′-(4″-hydroxyphenyl)ethyl} thiocarbamic acidp-(bis-2-chloroethylamino)phenyl ester (25).

A solution of the mustard 5 (0.2 g, 0.86 mmol) and methylamine (0.174 g,0.24 mL, 1.72 mmol) was slowly added to a refluxing solution ofpentafluorophenylchloro-thionoformate in toluene (3 mL) and the mixturewas heated under reflux for two hours. After this time, the reaction wascooled and concentrated in vacuo. The resultant product and tyrosinemethyl ester (0.336 g, 1.72 mmol) were then dissolved in anhydrousdimethylformamide (5 mL) and stirred under an inert atmosphere atambient temperature overnight. After this time the reaction wasconcentrated in vacuo and purified by column chromatography (silica gel,dichloromethane:ethyl acetate 95:5) to give thiocarbamate 25 as acolourless oil (0.30 g, 68%); [α]_(D) ²⁰ +53.4° (c 2.3, chloroform);R_(f) 0.42 (silica, dichloromethane:ethyl acetate 95:5); ν_(max) (KBrdisc) 3400, 1737, 1614, 1507, 1444, 1378, 1147/cm⁻¹; δ ¹H (400 MHz,CDCl₃) 3.16 (1H, dd, J 13.2 Hz, 4.8 Hz, CH ₂CH), 3.31 (1H, dd, J 14.3Hz, 5.5 Hz, CH ₂CH), 3.62 (4H, t, J 5.8 Hz, 2×NCH₂), 3.69 (4H, t, J 5.8Hz, 2×CH₂Cl), 3.77 (3H, s, OMe), 5.16 (1H, dd, J 13.2 Hz, 5.5 Hz, CH),5.56 (1H, br s, NH), 6.63 (2H, d, J 8.8 Hz, Ar), 6.77 (2H, d, J 7.9 Hz,Ar), 6.96 (2H, d, J 8.8 Hz, Ar), 6.99 (2H, d, J 7.9 Hz, Ar);

¹³C (100 MHz, CDCl₃); 36.1 (CH₂), 40.3 (CH₂), 52.6 (CH₃), 52.7 (CH₂),58.9 (CH), 112.0 (CH), 115.6 (CH), 123.5 (CH), 127.0 (C), 130.4 (CH),144.2 (C), 144.4 (C), 154.9 (C), 171.4 (C), 189.8.

Example 15

bis-(2-Chloroethyl)amino-4-hydroxyphenylaminomethanone (28).

di-(2-Chloroethyl)amino4-nitrophenoxymethanone (27).

Bis-(2-chloroethylamine) hydrochloride (5.6 mmol, 1 g) andp-nitrophenylchloroformate (5.4 mmol, 1 g) were dissolved indimethylformamide (15 mL) and triethylamine (11.5 mmol, 1.6 mL) wasadded slowly. The mixture was heated under reflux with stirring for 6hours before being concentrated in vacuo to give a brown oil.Purification by column chromatography (silica gel, dichloromethane)yielded carbamate 27 as a yellow oil (1.2 g, 70%). ν_(max) 1660, 1446,1378,1145/cm⁻¹; δ ¹H (400 MHz CDCl₃) 1.21–1.33 (4H, m, CH ₂), 3.38–3.47(4H, m, CH ₂), 7.51 (2H, d, J 5.7 Hz, 2×ArH), 8.32 (2H, d, J 5.7 Hz,2×ArH);

¹³C (100 MHz, CDCl₃) 30.8 (CH₂), 42.1 (CH₂), 44.4 (CH₂), 44.7 (CH₂),115.9 (2×CH), 131.2 (2×CH), 129.3 (C), 157.6 (C), 160.5 (C). m/z (Cl)273 (65%), 150 (50), 134 (100), 100 (10), 56 (25).

Example 16

bis-(2-Chloroethyl)amino-4-hydroxyphenylaminomethanone (28). 27 (860 mg,2.8 mmol) was dissolved in dimethylformamide (20 mL) and triethylamine(0.9 mL, 5.6 mmol) was added. The mixture was stirred at reflux for 30minutes and tyramine (0.76 mg, 5.6 mmol) was added. The mixture washeated under reflux for a further 6 hours and then concentrated invacuo. Purification by column chromatography (silica gel,dichloromethane then methanol) gave prodrug 28 as an orange/brown oil(571 mg, 67%). ν_(max) 3400, 1660, 1444, 1380, 1145/cm⁻¹; δ ¹H (400 MHz,CDCl₃) 2.61 (2H, t, J 3.72 Hz, 4,CH ₂Ar), 3.17–3.22 (10H, m, ArCH₂ CH ₂,2×CH ₂ CH ₂Cl), 6.67 (2H, d, J 5.7 Hz, 2×ArH), 6.9 (2H, d, J 5.7 Hz,2×ArH), 7.82 (1H, brs, NH);

¹³C (100 MHz, CDCl₃), 30.1 (CH₂), 43.1 (CH₂), 43.4 (CH₂), 44.2 (CH₂),46.2 (CH₂), 46.7 (CH₂), 115.9 (2×CH), 130 (2×CH), 130.1 (C), 155.6 (C),161.5 (C). m/z (Cl) 269 (87%), 224 (50), 138 (100), 121 (45), 108 (35).

Example 17

bis-(2-Chloroethyl)amino-4-hydroxyphenylaminomethanone (28). One potmethod. bis-(2-Chloroethyl)amine hydrochloride 26 (200 mg, 1.1 mmol) wasdissolved in dichloromethane (15 mL) and triethylamine (0.45 mL, 3.3mmol) was added. The mixture was stirred for 5 minutes at ambienttemperature and ethyl chloroformate or methyl chlorothioformate (1.32mmol) was added. The mixture was stirred until nobis-(2-chloroethyl)amine remained by TLC (ethyl acetate). Tyramine (300mg, 2.2 mmol) was added and the mixture was heated under reflux for 4hours. the mixture was allowed to cool, purified by dry flash columnchromatography (silica gel, dichloromethane 200 cm³) and concentrated invacuo to yield prodrug 28 as an orange/brown oil (234 mg, 67% for methylchlorothioformate and 229 mg, 64% for ethyl chloroformate).

Example 18

di-(2-Chloroethyl)amino4-hydroxyphenethyl(methyl)aminomethanone (29).bis-(2-Chloroethyl)amine hydrochloride 26 (200 mg, 1.1 mmol) wasdissolved in dichloromethane (15 mL) and triethylamine (0.45 mL, 3.3mmol) was added. The mixture was stirred for 5 minutes at ambienttemperature and methyl chlorothioformate (1.32 mmol) was added. Themixture was stirred until no bis-(2-chloroethyl)amine remained by TLC(ethyl acetate). N-mehty tyramine (322 mg, 2.1 mmol) was added and themixture was heated under reflux for 4 hours. The mixture was allowed tocool, purified by dry flash column chromatography (silica gel,dichloromethane 200 mL) and concentrated in vacuo to yield prodrug 29 asan orange/brown oil (68 mg, 18%). ν_(max) 3400, 1660, 1444, 1380,1145/cm⁻¹; δ ¹H (400 MHz, CDCl₃) 2.32 (3H, s, CH₃) 2.60 (2H, t, J 3.72Hz, CH ₂Ar), 3.17–3.22 (10H, m, ArCH_(2CH) ₂, 2×CH ₂ CH ₂Cl), 6.67 (2H,d, J 5.7 Hz, 2×ArH), 6.9 (2H, d, J 5.7 Hz, 2×ArH), 7.82 (1H, brs, NH);

¹³C (100 MHz, CDCl₃) 30.1 (CH₂), 43.3 (CH₂), 44.1 (CH₂), 44.2 (CH₂),45.2 (CH₂), 46.5 (CH₃) 46.6 (CH₂), 115.9 (2×CH), 130 (2×CH), 130.1 (C),155.6 (C), 161.5 (C). m/z (Cl) 283 (87%), 238 (50) 152 (100), 121 (45),108 (35).

Example 19

Methyl-2-di(2chloroethyl)aminocarbonylamino-3-(4-hydroxyphenyl)proponoate(30). bis-(2-Chloroethyl)amine hydrochloride 26 (200 mg, 1.1 mmol) wasdissolved in dichloromethane (15 mL) and triethylamine (0.45 mL, 3.3mmol) was added. The mixture was stirred for 5 minutes at ambienttemperature and methyl chlorothioformate (1.32 mmol) was added. Themixture was stirred until no bis-(2-chloroethyl)amine remained by TLC(ethyl acetate). Tyrosine methyl ester (411 mg, 2.1 mmol) was added andthe mixture was heated under reflux for 4 hours. The mixture was allowedto cool purified by dry flash column chromatography (silica gel,dichloromethane 200 mL) and concentrated in vacuo to yield prodrug 30 asan orange/brown oil (278 mg, 60%). ν_(max) 3400, 1740, 1660, 1444, 1380,1145/cm⁻¹; δ ¹H (400 MHz, CDCl₃) 2.32 (3H, s, CH₃) 2.60 (2H, t, J 3.72Hz, CH ₂Ar), 2.64 (3H, s, CH₃) 3.12–3.25 (9H, m, ArCH₂ CH, 2×CH ₂ CH₂Cl), 6.67 (2H, d, J 5.7 Hz, 2×ArH), 6.9 (2H, d, J 5.7 Hz, 2×ArH), 7.82(1H, brs, NH);.

¹³C (100 MHz, CDCl₃) 29.8 (CH₂), 43.3 (CH₂), 44.1 (CH₂), 44.2 (CH₂),45.2 (CH₂), 46.6 (CH₂), 47.1 (CH₃) 115.9 (2×CH), 130 (2×CH), 130.1 (C),155.6 (C), 160.3 (C) 161.5 (C). m/z (Cl) 242 (72%), 227 (45), 183 (75)152 (100), 121 (45), 108 (35).

Example 20

3-Acetyl-3,5,12-trihydroxy-1-[5-hydroxy-4-(4-hydroxyphenylaminocarbonylamino)-6-methylperhydro-2-pyranoloxy]-10-methoxy-(1S,3S)-1,2,3,4,6,11-hexaydro-6,11-naphthacenedione(32).

4-Hydroxyphenethylamino-4-nitrophenoxymethanone (31). Tyramine (1 g, 7.3mmol) and p-nitrophenylchloroformate (1.4 g, 7.3 mmol) were dissolved inanhydrous dichloromethane and heated under reflux for 2 hours. Thereaction mixture was allowed to cool, concentrated in vacuo and purifiedby dry flash column chromatography (silica, dichloromethane and thenethylacetate) to afford carbamate 31 as a pale yellow solid (2.2 g,97%); m.p. 157–159° C.; ν_(max) (KBr disc) 3400, 1658, 1440, 1380/cm−¹;δ ¹H (400 MHz, CDCl₃) 2.74 (2H, t, J 7.0 Hz, CH ₂Ar), 3.4 (2H, t, J 7.0Hz, CH₂) 6.76 (2H, d, J 8.5 Hz, 2×ArH), 7.06 (2H, d, J 8.5 Hz, 2×ArH),7.29 (2H, d, J 9.2 Hz, 2×ArH), 8.24 (2H, d, J 9.2 Hz, ArH);

¹³C (100 MHz, CDCl₃); 36.4 (CH₂), 44.2 (CH₂), 116.6 (2×CH), 123.7(2×CH), 126.4 (2×CH), 131.2 (2×CH), 131.3 (C) 146.9 (C), 156.0 (C),157.4 (C), 158.1 (C). m/z (Cl) 163 (25%), 139 (10), 107 (100) 65 (15).

3-Acetyl-3,5,12-trihydroxy-1-[5-hydroxy-4-(4-hydroxyphenylaminocarbonylamino)-6-methylperhydro-2-pyranoloxy]-10-methoxy-(1S,3S)-1,2,3,4,6,11-hexaydro-6,11-naphthacenedione(32).

Daunomyodin¹² (20 mg, 0.038 mmol) and carbamate 31 (15 mg, 0.049 mmol)were dissolved in dimethylformamide (1 mL) and diisopropylethylamine(7.5 μL, 0.042 mmol) was added. The flask was wrapped in tinfoil toexclude light and the mixture was stirred. for 3 hours. Diethyl ether (5mL) was added to give a red precipitate. The precipitate was collectedby filtering across a cotton wool plug. The solid was then washed offthe cotton wool using methanol (5 mL) and concentrated in vacuo to yieldprodrug 32 as a red oily solid (12 mg, 46%). ν_(max) 3400, 2720, 1740,1750, 1690, 1520, 1435, 1147/cm⁻¹; δ ¹H (400 MHz, CD₃OD) 1.25 (2H, d,J4.5 Hz, CH ₂), 1.32–1.4 (5H, m, 5′ CH₃and CH ₂CCOCH₃), 1.72–1.89 (2H,m, 2′CH₂), 2.35 (3H, s, COCH₃), 2.55 (2H, t, J 3.72 Hz, ArCH ₂CH₂N),3.18 (2H, t, J 3.72 Hz, ArCH₂ CH ₂N), 3.54–3.55 (1H, m, 4′CHOH), 3.71(1H, m, C(CHO)CH₂), 3.83 (3H, s, ArOCH₃), 3.92 (1H, brd, 1′CH), 4.25(1H, q, J 3.8 Hz, 5′CH), 5.31–5.33 (1H, m, 3′CH), 6.58 (2H, d, J 5.6 Hz,2×ArH), 6.91 (2H, d, J 5.6 Hz, 2×ArH), 7.27 (1H, t, J 3.3 Hz, ArH),7.55–7.57 (2H, m, 2×ArH);

¹³C (100 MHz, CD₃OD) 25.1 (CH₃), 31.2 (CH), 32.0 (CH₂), 33.9 (CH₂), 37.0(CH₂), 43.2 (CH₂), 44.2 (CH₂), 47.6 (CH) 56.2 (CH), 57.4 (CH), 69.1(CH), 74.3 (CH₃) 77.9 (C), 80.0 (CH₃) 102.7 (CH), 131.1 (3×CH), 131.8(CH), 135.9 (2×C), 136.2 (2×C), 136.3 (2×C), 137.4 (2×CH), 156.4 (C),157.2 (C), 157.7 (2×C), 160.6 (2×C), 187.6 (C), 187.9 (C), 214.1 (C);m/z (Cl) 383 (20%), 363 (100), 347 (15), 293 [urea linked tyramine to4-OH-5-Me-hexose(15)], 174 (10), 138 (10), 107 (10), 74 (10).

Example 21

In Vitro Tests

The stability of compounds according to the invention was assessed inthe following experiments.

Tests were run under cell-line conditions, that is, in RPMI 1640, fetalbovine serum, and L-glutamine at 37° C., and the percentagedecomposition was measured over time. The compounds tested representeddifferent linkages between the therapeutically active agent and the partof the molecule recognised by tyrosinase (i.e. carbamate linked,thiocarbamate linked, urea linked) and different therapeutically activeagents (i.e. normustine, phenol-mustard, daunomycin and taxolderivatives). As well as structure 1.1.2 given above, the structurestested were:

The results were as follows:

% decomposition over time Drug 1 hr 2 hrs 3 hrs 4 hrs 6 hrs 12 hrs 24hrs 48 hrs 1.1.2 0 0 0 0 0 0 0 0 1.1.5 0 0 0 0 0 0 0 0 1.1.1 7 44 61 74100 — — — 2.2.1 52 92 100 — — — — —

The results showed that compounds according to the invention having aurea linkage were more stable than compounds possessing a carbamatelinkage.

This test shows that the most stable linkage is a urea linkage. Theurea-linked prodrugs (1.1.2 and 1.1.5) showed no decomposition over atwo-day test period. The carbamate-linked prodrug (1.1.1) was lessstable in fetal bovine serum, and exhibited a half-life of approximately2.5 hrs. The thiocarbamate-linked prodrug (2.1.1) decomposed the mostrapidly, with a half-life of approximately 1.5 hrs.

Example 22

Oximetry

When tyrosinase substrates are oxidised according to the pathway inScheme 1, molecular oxygen is absorbed from the surrounding solution.The resulting oxygen depletion can be measured using an oxygen sensor,thereby oxygen uptake is a measure of the rate of tyrosinase oxidationof the prodrugs. Using this technique we were able to examine theoxidation of the prodrugs by tyrosinase (Table 2). The relative rates ofoxygen uptake were used to estimate the efficiency of the prodrugs toact as tyrosinase substrates. In order to obtain a quantitativecomparison of tyrosinase-catalysed oxidation of our prodrugs we alsoexamined the rate of oxygen uptake in the presence of the methyl esterof the natural substrate tyrosine (entry 14, table 2).

FIG. 1 is a graph of the oxidation rate of prodrugs by mushroomtyrosinase. FIG. 1 shows that prodrug 28bis-(2-chloroethylamino)-4-hydroxyphenylamino-methanone was an excellenttyrosinase substrate (R_(max)= 20 nanomoles/min). This is furtherdemonstrated by comparing oxygen depletion in the oximetry cuvettes ofprodrug 28 and tyrosine methyl ester over 400 seconds, which shows thatthe brief lag period is shortened in the case of the oxidation of theprodrug. (see FIG. 2). Prodrugs 9, 10 and 30 were also good substrates,exhibiting similar oxidation rates to tyrosine methyl ester. HetercatomIncorportation (oxygen or sulfur) to afford produrgs 21 and 22 resultedin slower oxidation. For example, the oxidation rate was only 7.5nanomoles/min for prodrug 21 and 22.

FIG. 2 is a graph of tyrosinase oxidation of tyrosine methyl ester andprodrug 28. The results of the oximetry study highlight the structuralproperties that diminish oxidation rate. For example, prodrug 16 was notoxidized by tryorinase within a 15 minutes incubation period. Weconclude that, shortening the dopamine chain lenght from two carbons toone carbon resulted in reduced affinity to the enzyme. This may be dueto increased steric hindrance close to the active site of the enzyme.Nitrogen methylation also resulted in a reduced rate of oxidation(prodrug 29), suggesting the importance of a primary or secondary aminefunction. Not surprisingly, the sterically hindered daunomycin-basedprodrug 32 was also a poor substrate with an R_(max) of only 5nanomles/min.

The results of the oximetry study highlight the structural propertiesthat diminish oxidation rate. For example, prodrug 16 was not oxidisedby tyrosinase within a 15 minutes incubation period. We conclude that,shortening the dopamine chain length from two carbons to one carbonresulted in reduced affinity to the enzyme. This may be due to increasedsteric hindrance close to the active site of the enzyme. Nitrogenmethylation also resulted in a reduced rate of oxidation (prodrug 29),suggesting the importance of a primary or secondary amine function. Notsurprisingly, the sterically hindered daunomycin-based prodrug 32 wasalso a poor substrate with an R_(max) of only 5 nanomoles/min.

-   1. A. M. Jordan, T. H. Khan, H. M. I. Osborn, A Photiou, P. A.    Riley, Biorg. Med. Chem, 1999, 7, 1775.-   2. M. H. Benn, A. M. Creighton, L. N. Owen, G. R. White, J. Chem.    Soc, 1961, 2365.-   3. T. Nakagawa, K. Ueno, M. Kashtwa, J. Wantanabe, Tetrahedron    Lett., 1194, 35, 1921.-   4. K. A. Jorgensen, A.-B. A. G. Ghattas, S. -O. Lawesson,    Tetrahedron, 1982, 38, 1163-   5. M. H. Benn, A. M. Creighton, L. N. Owen, G. R. White, J. Chem.    Soc., 1961, 2365-   6. T. Nakagawa, K. Ueno, M. Kashiwa, J. Wantanabe, Tetrahedron    Lett., 1994, 35, 1921-   7. M. Artico, C. J. Ross, Biochem. Pharmacol., 1968, 17, 893-   8. J. C. Clark, J. Chem. Soc. Perkin Trans. 1, 1976, 475-   9. E. P. Kyba, J. Amer. Chem. Soc., 1978, 100, 4555-   10. W. Kandatege, M. W. Sheldon, J. Ame. Chem. Soc., 1987, 109, 4036-   11. S. R. Padgette, H. H. Herman, J. H. Han, S. H. Pollock, S. W.    May, J. Med. Chem., 1984, 27, 1354

1. A compound having the formula (Ia), (Ib) or (Ic), or apharmaceutically acceptable salt thereof:

wherein R^(a) is hydrogen or —COOR^(b), R^(b) is hydrogen or C₁₋₆ alkyl;R^(d) and R^(e) independently represent hydrogen and hydroxy, R^(f) ishydrogen, C₁₋₄ alkyl or halogen, X is —CHOH—, —CH₂—oxygen or sulphur, mis zero or 1, W is oxygen or sulphur, and —ODrug is selected from:

a residue of taxol or gemcitabine; —NHDrug is selected from:

or a residue of daunomycin; and —N(Drug)₂ is:

and with the provisos (1) that where said compound has general formula(Ia) wherein m is zero, W represents oxygen, R^(d) is hydrogen and R^(e)is hydroxyl in the para position, and R^(a) represents —COOMe, —ODrugdoes not represent a residue of digoxin, and (2) that where saidcompound has general formula (Ib) wherein m is zero, W represents oxygenand R^(d) is hydrogen, R^(e) is hydroxyl in the para position.
 2. Acompound according to claim 1 wherein R^(d) represents m-hydroxy andR^(e) represents p-hydroxy.
 3. A compound according to claim 1 whereinR^(d) represents hydrogen and R^(e) represents p-hydroxy.
 4. A compoundaccording to claim 1 wherein m is zero.
 5. A compound according to claim1 wherein R^(f) is hydrogen.
 6. A compound according to claim 1 whereinW represents oxygen.
 7. A method of treating melanoma in a patient whichcomprises administering a compound having the formula (Ia), (Ib) or(Ic), or a pharmaceutically acceptable salt thereof:

wherein R^(a) is hydrogen or —COOR^(b), R^(b) is hydrogen or C₁₋₆ alkyl;R^(d) and R^(e) independently represent hydrogen and hydroxy, R^(f) ishydrogen, C₁₋₄ alkyl or halogen, X is —CHOH—, —CH2—oxygen or sulphur, mis zero or 1, W is oxygen or sulphur, and —ODrug is selected from:

a residue of taxol or gemcitabine; —NHDrug is selected from:

or a residue of daunomycin; and —N(Drug)₂ is: